| Field | Specification |
|---|---|
| Product Format | Frozen |
| Product Type | |
| Shipping | |
| Storage |
Overview
The Immortalized Immortalized Mouse EGFR Mutant M3-/- Epidermal Keratinocytes were derived from primary keratinocytes of newborn EGFR null mice. The primary keratinocytes were immortalized in subsequent cultures after passaging for an extended period.
Key elements and design rationale
- Model identity: Immortalized Mouse EGFR Mutant M3-/- Epidermal Keratinocytes is supplied as a tumor cell line derived from Mouse skin.
- Growth properties: Adherent, cobblestone
- Growth conditions: Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Thaw the cells with PriGrow XI (TM011) + 8% FBS + 60 µM CaCl₂ (added fresh each time) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 8% CO₂ for optimal attachment. After 24h, change media to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 8% CO₂ Steps to thaw the cells: 1. Thaw the cells in RT water until only a few ice crystals are left 2. Put the cells in 5 ml of RT PriGrow XI (TM011) + 8% FBS 3. Centrifuge the cells 1500 rpm for 5 min (need this step to remove inhibitors for attachment) 4. Resuspend the pellet in RT Prigrow XI (TM011) + 8% FBS + 1% P/S with freshly added 60 µM CaCl₂ and put the cells in a culture vessel at 37.0°C, 8% CO₂ 5. After 18h - 24h, change to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 8% CO₂ for growth of the cells.
- Product format: Frozen, BSL-2
This cell-based model is generally used in cancer biology, phenotype comparison, and response profiling studies. Donor/background information is available for contextual interpretation.
Biological background
The immortalized cells display similar phenotype to the primary keratinocytes including the cobblestone-shape morphology. The Immortalized Mouse EGFR Mutant M3-/- Epidermal Keratinocytes are non-tumorigenic. They also differentiate with increased calcium, maintain a relatively stable karyotype no more aberrant than cultured primary keratinocytes, and produce epidermis upon orthotopic grafting onto nude mouse hosts. The M3 muscarinic acetylcholine receptor is knocked out in these cells. Upon introduction of v-rasHa oncogene, the cells produce squamous cell carcinoma in orthotopic skin grafts. These cells are useful in studies focusing on epidermal keratinocyte biology and pathology. Donor/background information provided for this product: EGFR null mice, newborn.
Research relevance and current trends
- Cell-line models continue to be used for tumor phenotype comparison, pathway perturbation studies, and assay development in controlled in vitro systems.
- Engineered and subtype-defined tumor lines are often used to compare growth behavior, reporter output, and response patterns across matched experimental conditions.
- When metastatic or lineage features are described, investigators commonly interpret results alongside morphology, passage history, and culture environment.
Common research applications
- Cancer biology studies that compare proliferation-associated behavior, morphology, and pathway responses in vitro.
- Assay development for treatment response, reporter monitoring, or phenotype comparison under matched culture conditions.
- Side-by-side comparison of engineered versus parental background characteristics when relevant to the study design.
Changes in morphology, growth rate, viability, or reporter signal are typically interpreted together with passage history, culture matrix, and the specified growth conditions for the model.
Notes for experimental interpretation
- Morphology, doubling behavior, and reporter or marker output can shift with passage number, substrate choice, and medium composition; these variables should be recorded alongside experimental readouts.
- Matched controls such as parental cells, untreated cultures, or parallel cultures maintained under identical conditions help distinguish background effects from biology of interest.
Culture and product details
- Growth Conditions: Use of PriCoat™ T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Thaw the cells with PriGrow XI (TM011) + 8% FBS + 60 µM CaCl₂ (added fresh each time) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 8% CO₂ for optimal attachment. After 24h, change media to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 8% CO₂ Steps to thaw the cells: 1. Thaw the cells in RT water until only a few ice crystals are left 2. Put the cells in 5 ml of RT PriGrow XI (TM011) + 8% FBS 3. Centrifuge the cells 1500 rpm for 5 min (need this step to remove inhibitors for attachment) 4. Resuspend the pellet in RT Prigrow XI (TM011) + 8% FBS + 1% P/S with freshly added 60 µM CaCl₂ and put the cells in a culture vessel at 37.0°C, 8% CO₂ 5. After 18h - 24h, change to PriGrow XI (TM011) + 8% chelexed FBS + 1.4% FBS + 5 ng/ml rhFGF7 (Z100595) (freshly added) + 1% Penicillin/Streptomycin Solution (G255), 37.0°C, 8% CO₂ for growth of the cells.
- Seeding Density (cells/cm²): 20,000 - 30,000
- Thaw cells quickly in a 37°C water bath while agitating gently (maximum 2 minutes). The vial cap should be kept above the water level to minimize the risk of contamination.
- Decontaminate the vial by spraying and wiping the exterior of the vial with 70% ethanol. From this point onwards, all operations should be strictly carried out inside a biological safety cabinet using aseptic conditions.
- Transfer the cell suspension into a 15ml sterile conical tube containing 5ml of pre-warmed, complete growth media. Centrifuge cells at 125xg for 5-7 minutes.
- Aspirate the supernatant without disturbing the cell pellet. Re-suspend the cell pellet in the recommended pre-warmed, complete growth media and dispense into a T25 culture flask.
- Incubate the cells at the recommended conditions.
- Aspirate the culture media, and add 2-3ml of pre-warmed 0.25% Trypsin-EDTA to the culture vessel.
- Observe the cells under a microscope to confirm detachment (typically within 2-10 minutes). Cells that are difficult to detach can be put in 37°C, for several minutes to facilitate detachment.
- Neutralize Trypsin-EDTA by adding an equal volume of the complete growth media into the culture vessel.
- Transfer the culture suspension into a sterile centrifuge tube, and centrifuge at 125xg for 5 minutes. The actual centrifuge duration and speed may vary depending on the cell type.
- Aspirate the supernatant, and re-suspend the pellet with pre-warmed fresh complete growth media. Add appropriate aliquots of the cell suspension to new culture vessels, as desired.
- Incubate the cells at the recommended conditions.
How should I handle live cells once I receive them?
https://www.abmgood.com/immortalized-cells-documents.html
Following these guidelines will help ensure optimal cell viability and performance.
Why are these cells classified as biosafety level II?
What is your warranty or return policy?
Please refer to the following link for full information:
https://www.abmgood.com/terms
For additional questions, our Order team is happy to assist and can be reached at order@abmgood.com.
How many times can cells divide?
Primary cells have a limited lifespan and will undergo a finite number of population doublings before entering senescence. The exact number varies by cell type and culture conditions.
Immortalized cell lines are capable of extended or indefinite proliferation under proper culture conditions, although growth characteristics may vary between lines.
Do I need Applied Cell Extracellular Matrix (G422) if I am using PriCoat™ flasks?
Cell line sourcing and selection (species, tissue, and disease model matching) · Stable cell line engineering (overexpression, knockdown, knockout via CRISPR/Cas9, shRNA, sgRNA) · Reporter gene integration (GFP, RFP, luciferase, fluorescent/bioluminescent constructs) · Genome editing and knockin (point mutations, tagged endogenous proteins, conditional alleles) · Inducible expression systems (Tet-On/Off and regulatable constructs) · Drug resistance marker selection (puromycin, G418, hygromycin, and others) · Custom growth and media optimisation for specific assay requirements · Scale-up production for high-throughput screening campaigns · Authentication and QC services (STR profiling, mycoplasma testing, viability assessment). Talk to a Scientist or contact support@biohippo.com.
Hammiller, B. O., El-Abaseri, T. B., Dlugosz, A. A., & Hansen, L. A. (2015). A Method for the Immortalization of Newborn Mouse Skin Keratinocytes. Frontiers in oncology, 5, 177. https://doi.org/10.3389/fonc.2015.00177
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